title: control_M4_S17
description: Protocols: RNA was extracted (see nucleic acid extraction protocol) from small intestinal organoids (see growth protocol) treated with cytokines for 24 hours (see treatment protocol), a library was created (see nucleic acid library construction protocol) and sequenced single end 75 bp (see nucleic acid sequencing protocol) Crypts from small intestine were isolated according to a published protocol (Sato 2013). In summary, the small intestine of mouse was washed in ice-cold PBS and opened longitudinally. A microscopy cover slip was used to gently scrape of excess mucus and villus before the intestine was cut into 2-4 mm pieces. The fragments were resuspended in 30 mL ice-cold PBS and pipetted up and down with a 10 mL pipette. Supernatant was discarded and this step was repeated 5-10 times until the supernatant was clear. The fragments were incubated in 2 mM EDTA in PBS for small intestine at 4 C for 30 min with gentle rocking. The supernatant was removed, 20 mL of ice-cold PBS was added and the fragments were pipetted up and down. This washing step was repeated until the crypt fraction appeared as seen in a microscope. The 1-3 consecutive crypt fractions were passed through a 70 um cell strainer and collected into a FCS coated 50 mL tube. The crypts were spun down at 300 xg for 5 min and washed once in 10 mL ice cold PBS at 200 xg for 5 min. Approximately 200-500 crypts were resuspended in 50 uL Matrigel and kept at 4 C. 50 uL of the matrigel solution were added to the center of a pre-warmed 24 well plate and quickly transferred to an incubator at 37 degrees with 5 % CO2. After 5 min the pellet had solidified and 500 uL of basal culture medium was added and the plate was put back into the incubator. Basal culture medium consisted of advanced Dulbecco's modified Eagle medium - F12 supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM Glutamax, 1x N2 [ThermoFisher Scientific 100X, 17502048], 1x B-27 [ThermoFisher Scientific 50X, 17504044], and 1x N-acetyl-L-cysteine [Sigma, A7250]) and overlaid with ENR factors containing 50 ng/ml of murine EGF [Thermo Fisher Scientific, PMG8041], 20 % condition medium (CM) from a cell line producing R-Spondin (kind gift from Calvin Kuo, Stanford University School of Medicine, Stanford, CA, USA) and 10 % Noggin-CM (a kind gift from Hans Clevers, Hubrecht Institute, Utrecht, The Netherlands). The culture medium was replaced every 2-3 days. Organoids were passaged by disrupting them with strong mechanical pipetting, letting the solution cool on ice before centrifuging at 200 xg, 5 min at 4 C and resuspending in matrigel. Organoids were passages 2-3 times before being used in the experiment. References: Sato, Toshiro and Hans Clevers (2013). “Primary Mouse Small Intestinal Epithelial Cell Cultures”. In:Epithelial Cell Culture Protocols: Second Edition. Ed. by Scott H. Randell and M. Leslie Fulcher. Methods in Molecular Biology. Totowa, NJ: Humana Press, pp. 319-328.isbn: 978-1-62703-125-7.doi:10.1007/978-1-62703-125-7_19.527url:https://doi.org/10.1007/978-1-62703-125-7_19. 10 ng/mL cytokine (IL-13 Peprotech #210-13, IL-22 Peprotech #210-22, IFNG BioLegend #575306) was applied to wells 24 hours after splitting and the organoids where then incubated for 24 hours with cytokine. Three 24 well plate wells where combined together per sample for RNA extraction. RNA was extracted with Quick-RNA Microprep Kit according to the manufacturers instructions (Zymo, R1050). Library preparation was done with Lexogen SENSE mRNA kit
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-05-01T00:23:35Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-06-03T20:14:27Z
attributes.tag: External Id
attributes.value: SAMEA6872432
attributes.tag: INSDC center name
attributes.value: Dept. of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU)
attributes.tag: INSDC first public
attributes.value: 2021-05-01T00:23:35Z
attributes.tag: INSDC last update
attributes.value: 2020-06-03T20:14:27Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9182:control_M4_S17
attributes.tag: age
attributes.value: 7 to 8
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: epithelial cell of small intestine
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: organoid culture
attributes.tag: individual
attributes.value: C
attributes.tag: organism part
attributes.value: epithelium of small intestine
attributes.tag: sample name
attributes.value: E-MTAB-9182:control_M4_S17
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: C57BL/6